RESUMEN
Temozolomide (TMZ) is the leading therapeutic agent for combating Glioblastoma Multiforme (GBM). Nonetheless, the persistence of chemotherapy-resistant GBM cells remains an ongoing challenge, attributed to various factors, including the translesion synthesis (TLS) mechanism. TLS enables tumor cells to endure genomic damage by utilizing specialized DNA polymerases to bypass DNA lesions. Specifically, TLS polymerase Kappa (Polκ) has been implicated in facilitating DNA damage tolerance against TMZ-induced damage, contributing to a worse prognosis in GBM patients. To better understand the roles of Polκ in TMZ resistance, we conducted a comprehensive assessment of the cytotoxic, antiproliferative, antimetastatic, and genotoxic effects of TMZ on GBM (U251MG) wild-type (WTE) and TLS Polκ knockout (KO) cells, cultivated as three-dimensional (3D) tumor spheroids in vitro. Initial results revealed that TMZ: (i) induces reductions in GBM spheroid diameter (10-200 µM); (ii) demonstrates significant cytotoxicity (25-200 µM); (iii) exerts antiproliferative effects (≤25 µM) and promotes cell cycle arrest (G2/M phase) in Polκ KO spheroids when compared with WTE counterparts. Furthermore, Polκ KO spheroids exhibit elevated levels of cell death (Caspase 3/7) and display greater genotoxicity (53BP1) than WTE following TMZ exposure. Concerning antimetastatic effects, TMZ impedes invadopodia (3D invasion) more effectively in Polκ KO than in WTE spheroids. Collectively, the results suggest that TLS Polκ plays a vital role in the survival, cell death, genotoxicity, and metastatic potential of GBM spheroids in vitro when subjected to TMZ treatment. While the precise mechanisms underpinning this resistance remain elusive, TLS Polκ emerges as a potential therapeutic target for GBM patients.
Asunto(s)
ADN Polimerasa Dirigida por ADN , Resistencia a Antineoplásicos , Glioblastoma , Esferoides Celulares , Temozolomida , Humanos , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Glioblastoma/genética , Glioblastoma/enzimología , Temozolomida/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/genética , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Apoptosis/efectos de los fármacos , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/enzimología , Antineoplásicos Alquilantes/farmacologíaRESUMEN
Although several studies assess the biological effects of micro and titanium dioxide nanoparticles (TiO2 NPs), the literature shows controversial results regarding their effect on bone cell behavior. Studies on the effects of nanoparticles on mammalian cells on two-dimensional (2D) cell cultures display several disadvantages, such as changes in cell morphology, function, and metabolism and fewer cell-cell contacts. This highlights the need to explore the effects of TiO2 NPs in more complex 3D environments, to better mimic the bone microenvironment. This study aims to compare the differentiation and mineralized matrix production of human osteoblasts SAOS-2 in a monolayer or 3D models after exposure to different concentrations of TiO2 NPs. Nanoparticles were characterized, and their internalization and effects on the SAOS-2 monolayer and 3D spheroid cells were evaluated with morphological analysis. The mineralization of human osteoblasts upon exposure to TiO2 NPs was evaluated by alizarin red staining, demonstrating a dose-dependent increase in mineralized matrix in human primary osteoblasts and SAOS-2 both in the monolayer and 3D models. Furthermore, our results reveal that, after high exposure to TiO2 NPs, the dose-dependent increase in the bone mineralized matrix in the 3D cells model is higher than in the 2D culture, showing a promising model to test the effect on bone osteointegration.
RESUMEN
The demand for the development of three-dimensional (3D) cell culture models in both/either drug screening and/or toxicology is gradually magnified. Natural Products derived from plants are known as phytochemicals and serve as resources for novel drugs and cancer therapy. Typical examples include taxol analogs (i.e., paclitaxel and docetaxel), vinca alkaloids (i.e., vincristine, vinblastine), and camptothecin analogs (topotecan, irinotecan). Breast cancer is the most frequent malignancy in women, with a 70% chance of patients being cured; however, metastatic disease is not considered curable using currently available chemotherapeutic options. In addition, phytochemicals present promising options for overcoming chemotherapy-related problems, such as drug resistance and toxic effects on non-target tissues. In the toxicological evaluation of these natural compounds, 3D cell culture models are a powerful tool for studying their effects on different tissues and organs in similar environments and behave as if they are in vivo conditions. Considering that 3D cell cultures represent a valuable platform for identifying the biological features of tumor cells as well as for screening natural products with antitumoral activity, the present review aims to summarize the most common 3D cell culture methods, focusing on multicellular tumor spheroids (MCTS) of breast cancer cell lines used in the discovery of phytochemicals with anticancer properties in the last ten years.
Asunto(s)
Antineoplásicos , Productos Biológicos , Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/tratamiento farmacológico , Antineoplásicos/uso terapéutico , Paclitaxel , Esferoides Celulares/patología , Técnicas de Cultivo Tridimensional de Células , Fitoquímicos , Productos Biológicos/uso terapéutico , Línea Celular TumoralRESUMEN
Introduction: Cell-based assays using three-dimensional (3D) cell cultures may reflect the antitumor activity of compounds more accurately, since these models reproduce the tumor microenvironment better. Methods: Here, we report a comparative analysis of cell behavior in the two most widely employed methods for 3D spheroid culture, forced floating (Ultra-low Attachment, ULA, plates), and hanging drop (HD) methods, using the RT4 human bladder cancer cell line as a model. The morphology parameters and growth/metabolism of the spheroids generated were first characterized, using four different cell-seeding concentrations (0.5, 1.25, 2.5, and 3.75 × 104 cells/mL), and then, subjected to drug resistance evaluation. Results: Both methods generated spheroids with a smooth surface and round shape in a spheroidization time of about 48 h, regardless of the cell-seeding concentration used. Reduced cell growth and metabolism was observed in 3D cultures compared to two-dimensional (2D) cultures. The optimal range of spheroid diameter (300-500 µm) was obtained using cultures initiated with 0.5 and 1.25 × 104 cells/mL for the ULA method and 2.5 and 3.75 × 104 cells/mL for the HD method. RT4 cells cultured under 3D conditions also exhibited a higher resistance to doxorubicin (IC50 of 1.00 and 0.83 µg/mL for the ULA and HD methods, respectively) compared to 2D cultures (IC50 ranging from 0.39 to 0.43). Conclusions: Comparing the results, we concluded that the forced floating method using ULA plates was considered more suitable and straightforward to generate RT4 spheroids for drug screening/cytotoxicity assays. The results presented here also contribute to the improvement in the standardization of the 3D cultures required for widespread application.
RESUMEN
Trichomonas tenax is considered a commensal organism found under poor oral hygiene conditions. T. tenax presents morphological similarities with T. vaginalis, and there are doubts concerning whether this protist is a parasite and whether it is a genetic variant of T. vaginalis. This study aimed to investigate the capacity of T. tenax to cause mammalian cell damage and compare its cytotoxicity with that of T. vaginalis. Protozoan-host cell interaction assays were performed with Madin-Darby canine kidney, HeLa, and gum cells and 3D spheroids, which were examined by scanning electron and transmission electron microscopy. Cellular viability experiments were also performed. T. tenax attached and had different forms when interacting with mammalian cells and caused damage with time-dependent host-cell viability. We observed that T. tenax produced plasma membrane projections and phagocytosed portions of the mammalian cells. In addition, T. tenax caused membrane blebbing and apoptotic bodies in HeLa cells, thus inducing cell death. Spheroids were also used in interaction assays with T. tenax and they were damaged by these cells. This study shows that T. tenax fulfills the requisites of a parasite, causing damage to different mammalian cells and behaving similarly to T. vaginalis when in contact with target cells in vitro.